Quiescence of Limbal Epithelial Stem Cells Summary The stem cells (SCs) of the corneal epithelium located in the limbal ?palisades of Vogt? are the ultimate source of maintaining corneal epithelial homeostasis. Clinically, loss of limbal SCs or dysfunction of the limbal niche leads to corneal blindness due to limbal SC deficiency (LSCD). During the last funding period, we successfully isolated and expanded limbal niche cells (NC) and established in vitro niches by SC-NC reunion in 3D Matrigel or HC-HA/PTX3, i.e., a novel matrix purified from amniotic membrane. Using these in vitro models, we have proven the hypothesis that maintenance of close SC-NC contact is crucial for preventing SCs from adopting corneal fate decision and maintaining SC self-renewal and quiescence through a balancing act between Wnt and BMP signaling. Herein, we propose to explore the signaling mechanism wherein SC quiescence is controlled by delineating how HC-HA/PTX3 transmits CD44-mediated signaling to maintain the NC phenotype and activate BMP signaling (Aim 1) and how PCP signaling reinforces BMP signaling in NCs to augment SC quiescence (Aim 2). Furthermore, we will determine how Notch signaling is established between SCs and NCs through the polar expression of Notch ligands and receptors under the influence of PCP signaling so as to transmit BMP signaling from NCs to SCs to achieve SC quiescence (Aim 3). In 3D Matrigel, noggin suppresses BMP signaling to allow full activation of Wnt signaling in order to promote SC self-renewal. In HC-HA/PTX3, we will determine whether noggin acts differently from in 3D Matrigel by inducing an ?alert? stage of SC self-renewal without affecting pre-existing BMP and PCP signaling by upregulating NKD1 that continues to suppress Wnt signaling (Aim 4). Successful completion of the above Aims will shed new light on the signaling mechanism wherein SC quiescence and self-renewal are maintained by close contact with NCs. Consequently, we will learn a better strategy to maintain the SC pool so as to ensure homeostasis of the corneal epithelium. This new knowledge will also help us devise a new tissue engineering strategy for the corneal epithelium by successful recapitulation of the regulatory mechanism executed by close interaction with NCs. Future studies of how such signaling might be altered by pathogenic factors derived from non-resolving inflammation will help us unravel the pathogenesis and potential new therapies of LSCD. Collectively, these studies may one day help us realize the considerable promise held by adult SCs in treating a number of diseases in the body.